PROJECT SUMMARY Nearly 20% of all cancers contain mutations in subunits from the SWI/SNF family of Nucleosome (Nuc) remodeling complexes. The SWI/SNF family regulates local genome access by pumping DNA around histone octamers, thus ?sliding? or remodeling Nucs within chromatin. Recurrent somatic mutations in SWI/SNF subunits are observed in multiple cancers, supporting a driver role in tumorigenesis. The mutated complexes are desirable therapeutic targets, since removing their ATPase activity promotes cancer cell death but spares normal cells. This phenomenon is known as synthetic lethality and identifying inhibitors to exploit it may lead to drugs with cancer specificity. In Phase I, EpiCypher developed EpiDyneTM, recombinant nucleosome remodeling substrates for SWI/SNF family enzymes. Our assay design utilized a GATC motif within a defined Nucleosome positioning sequence (NPS), where the positioned histone octamer blocks access to the motif. Upon nucleosome remodeling, the GATC motif is exposed and can be subsequently cleaved or labeled as an assay readout. The primary goals of our Phase II research program are to evolve EpiDyne toward non-radioactive HTS readouts as we begin to scale-up and validate our SWI/SNF inhibitor screening platform for commercial release. In Aim 1, we will develop novel specialized EpiDyne nucleosomes (epiNucs) compatible with multiple non-radioactive HTS readouts (e.g. Fluorescence polarization [FP] or Alpha). In addition, we will engineer epiNucs containing acetylation on histone H3, a post-translational modification (PTM) that interacts with a bromodomain on SMARCA2/4 (the SWI/SNF complex ATPase subunit) and enhances remodeling activity in vitro / in vivo. The use of physiological substrates is preferable for biochemical inhibitor assays as these are more likely to identify compounds with in vivo activity. Thus, we hypothesize that acetylated epiNucs may not only enhance enzyme activity (i.e. increase assay window) but may lead to the identification of more target specific inhibitors (vs. unmodified epiNucs). In Aim 2, we will develop methods for the manufacturing of SMARCA2 and SMARCA4 containing SWI/SNF remodeling complexes (not commercially available). Using these remodeling complexes, we will optimize HTS assays using Alpha or FP readouts; the development of orthogonal HTS platforms will allow users to perform both primary screens and hit counterscreening. In Aim 3, we will scale-up manufacturing of epiNucs (unmodified and acetylated). Finally, we will perform a series of pilot screens (4,500 compound library; in collaboration with Jim Bruenig at BeanTown Biotech) using our optimized SMARCA2 or SMARCA4 HTS assays. Given the remarkable disease prevalence of dysfunctional nucleosome remodeling, the HTS platform(s) we develop will have a profound impact on the identification of new therapeutics for diverse human diseases, most notably cancers with poor prognosis.